Nanostructure surface coated medical implants and methods of using the same

ABSTRACT

Compositions including a surface or film comprising nanofibers, nanotubes or microwells comprising a bioactive agent for elution to the surrounding tissue upon placement of the composition in a subject are disclosed. The compositions are useful in medical implants and methods of treating a patient in need of an implant, including orthopedic implants, dental implants, cardiovascular implants, neurological implants, neurovascular implants, gastrointestinal implants, muscular implants, and ocular implants.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.12/530,015, filed on Mar. 1, 2011, issued as U.S. Pat. No. 9,775,932,which is a 371 of International Patent Application No.PCT/US2008/057261, filed on Mar. 17, 2008, which claims the benefit ofU.S. Provisional Application No. 60/895,306 filed on Mar. 16, 2007, andU.S. Provisional Application No. 60/911,424, filed on Apr. 12, 2007,which applications are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

Total joint replacement is an effective treatment for relieving pain andrestoring function for patients with damaged or degenerative joints.Approximately 500,000 total hip and knee replacements are performed eachyear in the United States. Although many of the outcomes are successful,there are still significant problems with implant loosening and failure.In fact, 25% of hip replacement surgeries were revisions due to previousimplant failure. Surgery to replace these failures is more difficult andcostly to perform and has a poorer outcome than the original jointreplacement surgery. If fixation is not sufficient, loosening andosteolysis of the implant can occur. To overcome this problem, it isthought that bone implant materials need to stimulate rapid boneregeneration in order to fill in deficient bone and fix the implantfirmly with the adjacent bone. The material surface must be able torecruit bone forming cells, such as osteoblasts, such that they cancolonize and synthesize new bone tissue.

In order to design better implant materials, it is important tounderstand the events at the bone-material interface. As mentionedearlier, one of the important challenges is to induce bone growth on theimplant surface. The level of bone growth depends on the surfacecharacteristics of the implant. The first event that occurs after theimplantation of a biomaterial is the adsorption of proteins from bloodand other tissue fluids. Primarily, a hematoma, swelling filled withblood due to a break in the blood vessel, is present between the implantand bone. Cytokines and growth factors stimulate the recruitment ofmesenchymal cells which differentiate into osteoblast that areresponsible for bone formation. Over time, woven bone matures intolamellar bone which further strengthens the bone-implant interface.Thus, the surface properties play a critical role in long term stabilityand functionality of the implant.

In an attempt to enhance the stability of endosseous implants, a largenumber of implant materials and designs have been used. In addition tocement-based prosthetics, much attention in recent years has turned tomicrointerlocked implants, which have microporous surfaces to allow forthe ingrowth of bone. Early work using oxide ceramics showed that aminimum interconnected pore diameter of approximately 100 μm was neededfor adequate bone ingrowth (Hulbert et al., J Biomed Mater Res 1972;6(5):347-74). It was thought that smaller pore sizes allowed incompletemineralization of the infiltrating tissue. Subsequent use of metallicimplants showed bone ingrowth with pore sizes between 50 and 400 μm(Bobyn et al., Clin Orthop Relat. Res 1980(150):263-70). However, recentstudies have revealed the possibility that much smaller pores may allowbone ingrowth when presented at high density within metal-oxidesubstrates. For example, nanoporous Ca—P coatings on implants have shownapposition of human bone growth within 2-3 weeks post surgery (Lee etal., J Biomed Mater Res 2001; 55(3):360-7). Osteoblasts cultured onceramics of different nm-scale textures also exhibit alteredmorphologies and growth rates (Boyan et al., Biomaterials 1996,17(2):137-46; Popat et al., J Orthop Res 2006, 24(4):619-27; Popat etal., Biomaterials 2005, 26(22):4516-22; Swan et al., Biomaterials 2005,26(14):1969-76; Swan et al., J Biomed Mater Res A 2005, 72(3):288-95;Webster et al., Biomaterials 2004, 25(19):4731-9; Webster et al., JBiomed Mater Res A 2003, 67(3):975-80; Webster et al., Biomaterials2000, 21(17):1803-10). Nonetheless, there are several problems relatedto dissolution of nanoscale coatings over time, and cracking andseparation from the metallic substrate (Bauer et al., Clin Orthop RelatRes 1994, (298):11-8; and Bloebaum et al., Clin Orthop Relat Res 1994,(298):19-26). These studies point to the importance of developing morerobust and flexible nanoscale architectures to enhance the apposition ofbone from existing bone surfaces and stimulate new bone formation.

This invention described below addresses these needs, as well as others.

SUMMARY OF THE INVENTION

The present invention provides compositions including a surface or filmcomprising nanofibers, nanotubes, or microwells, comprising a bioactiveagent for elution to the surrounding tissue upon placement in a subject.The compositions are useful as medical implants, including orthopedicimplants, dental implants, cardiovascular implants, neurologicalimplants, neurovascular implants, gastrointestinal implants, muscularimplants, and ocular implants. The present invention also providesmethods of treating a patient in need of such an implant.

The present invention provides a medical implant including a surface orfilm comprising a plurality of nanofibers, nanotubes, or microwells,where said nanofibers, nanotubes, or microwells comprise a bioactiveagent for elution to the surrounding tissue upon placement in a subject.In some embodiments, the medical implant is an orthopedic implant, adental implant, a cardiovascular implant, a neurological implant, aneurovascular implant, a gastrointestinal implant, a muscular implant,or an ocular implant. In some embodiments, the medical implant is apatch for localized delivery of said bioactive agent to a soft tissue.In some embodiments, the surface or film expands or unfurls in thepresence of a hydrating liquid. In some embodiments, the surface or filmfurther includes cells, such as a stem cell, a retinal progenitor cell,a cardiac progenitor cell, an osteoprogenitor cell, or a neuronalprogenitor cell.

In some embodiments, the at least one of said plurality of nanofibers,nanotubes, or microwells further comprises an erodible capping film toprovide for delayed elution of said bioactive agent. In someembodiments, the surface or film comprises a plurality of at least twoof nanofibers, nanotubes, and microwells, wherein one of saidnanofibers, nanotubes, and microwells further comprises an erodiblecapping film to provide for delayed elution of said bioactive agent.

In some embodiments, the surface or film is comprised ofpoly(DL-lactide-co-glycolide) (PLGA), poly(DL-lactide-co-ε-caprolactone)(DLPLCL), poly(ε-caprolactone) (PCL), collogen, gelatin, agarose,poly(methyl methacrylate), galatin/ε-caprolactone, collagen-GAG,collagen, fibrin, PLA, PGA, PLA-PGA co-polymers, poly(anhydrides),poly(hydroxy acids), poly(ortho esters), poly(propylfumerates),poly(caprolactones), poly(hydroxyvalerate), polyamides, polyamino acids,polyacetals, biodegradable polycyanoacrylates, biodegradablepolyurethanes and polysaccharides, polypyrrole, polyanilines,polythiophene, polystyrene, polyesters, non-biodegradable polyurethanes,polyureas, poly(ethylene vinyl acetate), polypropylene,polymethacrylate, polyethylene, polycarbonates, poly(ethylene oxide),co-polymers of the above, mixtures of the above, and adducts of theabove, or combinations thereof.

In some embodiments, the surface or film is comprised of silicon,titania, zirconia, cobalt-chromium, alumina, silica, barium aluminate,barium titanate, iron oxide, and zinc oxide, nitinol, elastinite,tantalum, elgiloy, phynox, Ti6Al4V, CoCr, TiC, TiN, L605, 316, MP35N,MP20N, stainless steel alloy, 316L stainless steel alloy, 304 stainlesssteel alloy, or combinations thereof.

In some embodiments, the surface or film further includes a covalentlyattached bioactive agent. In some embodiments, the nanofibers,nanotubes, or microwells further include an agent to facilitate celladhesion and cell growth selected from the group consisting of laminin,fibrin, fibronectin, proteoglycans, glycoproteins, glycosaminoglycans,chemotactic agents, and growth factors. In some embodiments, thebioactive agent is selected from a polypeptide, growth factor, a steroidagent, an antibody therapy, an antibody fragment, a DNA, an RNA, andsiRNA, an antimicrobial agent, an antibiotic, an antiretroviral drug, ananti-inflammatory compound, an antitumor agent, anti-angiogeneic agent,and a chemotherapeutic agent.

In some embodiments, the nanofibers or nanotubes range in length fromabout 1 μm to about 500 μm, such as from about 1 μm to about 70 μm. Insome embodiments, the nanofibers or nanotubes range in diameter fromabout 3 nm to about 300 nm. In some embodiments, the nanotubes have apore diameter range from about 3 nm to about 250 nm. In someembodiments, the surface or film comprises nanofibers at a densitygreater than 100,000,000 nanofibers per square centimeter. In someembodiments, the surface or film comprises nanotubes at a densitygreater than 10,000,000 nanotubes per square centimeter. In someembodiments, the surface or film comprises nanofibers at a densitygreater than 25,000,000 nanofibers per square centimeter, wherein saiddensity provides for an extracellular matrix compatible tissue adhesive.In some embodiments, the surface or film comprises nanotubes at adensity greater than 25,000,000 nanotubes per square centimeter, whereinsaid density provides for an extracellular matrix compatible tissueadhesive.

In some embodiments, the said surface or film ranges in thickness fromabout 1 μm to about 2.5 mm, such as from about 1 μm to about 750 μm,including from about 1 μm to about 200 μm, and from about 1 μm to about150 μm.

In some embodiments, the microwells range in diameter from about 1 μm toabout 150 μm. In some embodiments, the microwells range in diameter fromabout 1 μm to about 150 μm. In some embodiments, the surface or filmcomprises microwells at a density greater than 150,000 microwells persquare centimeter.

The present invention also provides a method of treating a patient inneed of a medical implant, by placing a medical implant into thepatient, wherein the medical implant comprises a surface or filmcomprising a plurality of nanofibers, nanotubes, or microwells, wheresaid nanofibers, nanotubes, or microwells comprise a bioactive agent forelution to the surrounding tissue upon placement in a subject. In someembodiments, the medical implant is an orthopedic implant, a dentalimplant, a cardiovascular implant, a neurological implant, aneurovascular implant, a gastrointestinal implant, a muscular implant,or an ocular implant. In some embodiments, the medical implant is apatch for localized delivery of said bioactive agent to a soft tissue.In some embodiments, the surface or film expands or unfurls in thepresence of a hydrating liquid. In some embodiments, the surface or filmfurther includes cells, such as a stem cell, a retinal progenitor cell,a cardiac progenitor cell, an osteoprogenitor cell, or a neuronalprogenitor cell.

In some embodiments, the at least one of said plurality of nanofibers,nanotubes, or microwells further comprises an erodible capping film toprovide for delayed elution of said bioactive agent. In someembodiments, the surface or film comprises a plurality of at least twoof nanofibers, nanotubes, and microwells, wherein one of saidnanofibers, nanotubes, and microwells further comprises an erodiblecapping film to provide for delayed elution of said bioactive agent.

In some embodiments, the surface or film is comprised ofpoly(DL-lactide-co-glycolide) (PLGA), poly(DL-lactide-co-ε-caprolactone)(DLPLCL), poly(ε-caprolactone) (PCL), collogen, gelatin, agarose,poly(methyl methacrylate), galatin/ε-caprolactone, collagen-GAG,collagen, fibrin, PLA, PGA, PLA-PGA co-polymers, poly(anhydrides),poly(hydroxy acids), poly(ortho esters), poly(propylfumerates),poly(caprolactones), poly(hydroxyvalerate), polyamides, polyamino acids,polyacetals, biodegradable polycyanoacrylates, biodegradablepolyurethanes and polysaccharides, polypyrrole, polyanilines,polythiophene, polystyrene, polyesters, non-biodegradable polyurethanes,polyureas, poly(ethylene vinyl acetate), polypropylene,polymethacrylate, polyethylene, polycarbonates, poly(ethylene oxide),co-polymers of the above, mixtures of the above, and adducts of theabove, or combinations thereof.

In some embodiments, the surface or film is comprised of silicon,titania, zirconia, cobalt-chromium, alumina, silica, barium aluminate,barium titanate, iron oxide, and zinc oxide, nitinol, elastinite,tantalum, elgiloy, phynox, Ti6Al4V, CoCr, TiC, TiN, L605, 316, MP35N,MP20N, stainless steel alloy, 316L stainless steel alloy, 304 stainlesssteel alloy, or combinations thereof.

In some embodiments, the surface or film further includes a covalentlyattached bioactive agent. In some embodiments, the nanofibers,nanotubes, or microwells further include an agent to facilitate celladhesion and cell growth selected from the group consisting of laminin,fibrin, fibronectin, proteoglycans, glycoproteins, glycosaminoglycans,chemotactic agents, and growth factors. In some embodiments, thebioactive agent is selected from a polypeptide, growth factor, a steroidagent, an antibody therapy, an antibody fragment, a DNA, an RNA, andsiRNA, an antimicrobial agent, an antibiotic, an antiretroviral drug, ananti-inflammatory compound, an antitumor agent, anti-angiogeneic agent,and a chemotherapeutic agent.

In some embodiments, the nanofibers or nanotubes range in length fromabout 1 μm to about 500 μm, such as from about 1 μm to about 70 μm. Insome embodiments, the nanofibers or nanotubes range in diameter fromabout 3 nm to about 300 nm. In some embodiments, the nanotubes have apore diameter range from about 3 nm to about 250 nm. In someembodiments, the surface or film comprises nanofibers at a densitygreater than 100,000,000 nanofibers per square centimeter. In someembodiments, the surface or film comprises nanotubes at a densitygreater than 10,000,000 nanotubes per square centimeter. In someembodiments, the surface or film comprises nanofibers at a densitygreater than 25,000,000 nanofibers per square centimeter, wherein saiddensity provides for an extracellular matrix compatible tissue adhesive.In some embodiments, the surface or film comprises nanotubes at adensity greater than 25,000,000 nanotubes per square centimeter, whereinsaid density provides for an extracellular matrix compatible tissueadhesive.

In some embodiments, the said surface or film ranges in thickness fromabout 1 μm to about 2.5 mm, such as from about 1 μm to about 750 μm,including from about 1 μm to about 200 μm, and from about 1 μm to about150 μm.

In some embodiments, the microwells range in diameter from about 1 μm toabout 150 μm. In some embodiments, the microwells range in diameter fromabout 1 μm to about 150 μm. In some embodiments, the surface or filmcomprises microwells at a density greater than 150,000 microwells persquare centimeter.

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the invention as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures:

FIG. 1 is a series of SEM images of titania nanotubular surfaces. Theleft panel shows a cross-sectional view of a mechanically fracturedsample; the center panel is a top view of nanotubular surface; and theright panel is a high magnification top view of nanotubular surface. Thenanotubes are approximately 80 nm in diameter and 400 nm long.

FIG. 2, panel a, shows marrow stromal cell adhesion and proliferation onpolystyrene, titanium and nanotubular surfaces for up to 7 days ofculture, nanotubular surfaces show approx. 40% more cell proliferationafter 7 days of culture compared to titanium surface (p<0.05); panel bshows cell viability measured as absorbance using MTT assay after 4 daysfor cell culture on polystyrene, titanium and nanotubular surface.

FIG. 3 shows fluorescence microscopy images (10×) of live marrow stromalcells stained with calcein on (panel a) titanium and (panel b)nanotubular surfaces; the cells seem to form clusters on nanotubularsurface which is absent on titanium surfaces.

FIG. 4 shows a series of SEM images of marrow stromal cells on titaniumand nanotubular surfaces for up to 7 days of culture. Cells showspherical morphology on titanium (panel a) compared to spreadingmorphology on nanotubular surface (panel b) after 1 day of culture.After 4 days of culture, cells still show spherical morphology ontitanium surface (panel c) compared to spreading and clusteringmorphology on nanotubular surface (panel d). After 7 days of culture,some of the cells on titanium seem to be spreading (panel e), howeverthe cells show high degree of spreading and have started communicatingon nanotubular surface (panel f). High magnification SEM image after 7days of culture (panel g) on nanotubular surface shows that cellextensions are protruding into the nanotubular architecture.

FIG. 5, panel a, is a graph showing ALP activity measured for up to 3weeks of culture of marrow stromal cells on titanium and nanotubularsurface after providing complete media, data normalized with totalprotein content to account for the effect of different number of cellson each surface, ALP activity is approx. 50% higher after 3 weeks ofculture on nanotubular surfaces (p<0.05); panel b is a graph showingCalcium concentration measured for up to 3 weeks of culture of marrowstromal cells on titanium and nanotubular surface, data normalized withtotal protein content to account for the effect of different number ofcells on each surface, calcium content is approx. 50% higher after 3weeks on nanotubular surface (p<0.05).

FIG. 6 is a series of SEM images of MSCs on nanotubular surfaces for upto 3 weeks of culture. Formation of cell clusters (panel a) anddeposition of granular material (panel b) on nanotubular surfaces after1 week; After 2 weeks, the surface is almost all covered by cells (panelc) and the nanotubes are further filled with matrix (panel d). After 3weeks, the entire surface is covered with well spread cells (panel e)and the nanotubes are almost completely filled with matrix constituents(panel f).

FIG. 7 is a series of images of histological analysis of tissue (panela) control—normal healthy tissue; (panel b) surrounding titaniumimplant; (panel c) surrounding nanotubular implant; results indicate nofibrous scar tissue formation for both titanium and nanotubular implantand the tissues are very similar to the control tissue, dotted lineshows where the implant was in contact with tissues.

FIG. 8 is graph showing the loading efficiencies of bovine serum albumin(BSA) and lysozyme (LYS) in nanotubes.

FIG. 9 is a series of graphs showing the fraction of total proteinreleased from nanotubes filled with 200 mg (Panel A), 400 mg (Panel B)and 800 mg (Panel C) of BSA and 200 mg (Panel D), 400 mg (Panel E) and800 mg (Panel F) of LYS. The time point at which all the protein isreleased is indicated by dotted line. Concentrations at these timepoints are significant different then those for time points before,however not significantly different then the time points after, p<0.05,n=3

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions including a surface or filmcomprising nanofibers, nanotubes, or microwells, comprising a bioactiveagent for elution to the surrounding tissue upon placement in a subject.The compositions are useful as medical implants, including orthopedicimplants, dental implants, cardiovascular implants, neurologicalimplants, neurovascular implants, gastrointestinal implants, muscularimplants, and ocular implants. The present invention also providesmethods of treating a patient in need of such an implant.

Before the present Invention described, it is to be understood that thisinvention is not limited to particular embodiments described, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting, since the scope of the presentinvention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupercedes any disclosure of an incorporated publication to the extentthere is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the compound”includes reference to one or more compounds and equivalents thereofknown to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

INTRODUCTION

The present invention is based on the observation that nanotubularsurfaces provide a favorable template for bone cell growth anddifferentiation and supported higher cell adhesion, proliferation andviability, while not causing adverse immune response under in vivoconditions. The biocompatibility of metal-oxides has already been provenas the materials have current clinical applications in orthopedicprostheses and dental implants. The inventors have found that osteoblastactivity can be significantly enhanced using controllednanotopographies. Therefore, incorporation of such nanoarchitectures onmedical implant surfaces further facilitates the culture and maintenanceof differentiated cell states, and promotes long-term osseointegration.

The inventors also found that these nanotubes can be optionally loadedwith drugs or biological agents such as proteins. Moreover, the releaseor elution of the drugs or biological agents from the nanotubes can becontrolled by varying the tube length, diameter and wall thickness. Bychanging the nanotube diameter, wall thickness and length, the releasekinetics can be altered for specific drugs in order to achieve sustainedrelease of the drug over a period of time. Thus, these nanotubularsurfaces have various potential applications, specifically for implantswhere faster integration is desired along with controlled release ofdrugs such as antibiotics or growth factors.

The invention is now described in greater detail.

Methods and Compositions

As noted above, the present invention provides compositions including asurface or film comprising a plurality of nanofibers, nanotubes, ormicrowells for use in treating a subject in need of a medical implant.In some embodiments, the nanotubes can be optionally be loaded with abioactive agent for elution to the surrounding tissue upon placement ofthe implant in a subject. Exemplary medical implants include, but arenot limited to, an orthopedic implant, a dental implant, acardiovascular implant, a neurological implant, a neurovascular implant,a gastrointestinal implant, a muscular implant, an ocular implant, andthe like. In some embodiments, the surface or film is a patch that canbe used for localized delivery of the bioactive agent to a soft tissue,such as liver, kidney, gastrointestinal tract, pancreas, prostate,colon, and the like. Exemplary bioactive agent include, but are notlimited to, polypeptides, nucleic acids, such as DNA, RNA, and siRNA,growth factors, steroid agents, antibody therapies, antimicrobialagents, antibiotics, antiretroviral drugs, anti-inflammatory compounds,antitumor agents, anti-angiogeneic agents, and chemotherapeutic agents.In certain embodiments, the surface or film further includes acovalently attached bioactive agent. In some embodiments, surface orfilm further includes cells, such as stem cells, retinal progenitorcells, cardiac progenitor cells, osteoprogenitor cells, neuronalprogenitor cells, and the like.

In some embodiments, the surface or film expands or unfurls in thepresence of a hydrating liquid, such as water present in an insertionsite of a subject. By “expands” is meant that surface or film becomeslarger in size or volume as a result surrounding liquid hydrating thesurface or film. By “unfurl” is meant that the surface or film isunrolled, unfolded, or spread out as a result surrounding liquidhydrating the surface or film

Exemplary surfaces and films can be fabricated from a variety ofsuitable materials that provide the ability to form the desiredplurality of nanotubes, nanofibers, and microwells. Exemplary materialsinclude, but are not limited to, biodegradable or bioerodible polymer,such as poly(DL-lactide-co-glycolide) (PLGA),poly(DL-lactide-co-ε-caprolactone) (DLPLCL), or poly(ε-caprolactone)(PCL), as well as natural biodegradable polymers, such as collogen,gelatin, agarose, and the like. PLGA is a bulk-eroding copolymer ofpolylactide (PLA) and polyglycolide (PGA), where the ingress of water isfaster than the rate of degradation. In this case, degradation takesplace throughout the whole of the polymer sample, and proceeds until acritical molecular weight is reached, at which point degradationproducts become small enough to be solubilized. At this point, thestructure starts to become significantly more porous and hydrated. Thecombination of fast-resorbing PGA and slow-resorbing PLA allows PLGAcopolymers to have a resorption rate of approximately 6 weeks.Fast-resorbing PLGA polymers display high shrinkage, which may notpresent a stable substrate for cells to lay down extracellular matrix.In addition, the production of acidic degradation species byfast-resorbing polymers can compromise tissue repair.

In addition, the surface or film can be fabricated from a variety ofsuitable metal oxides selected from the group consisting of alumina,titania, Ti6Al4V, nickel, zirconia, cobalt-chromium (CoCr), alumina,silica, barium aluminate, barium titanate, iron oxide, and zinc oxide,as well as shape memory alloys, such as nitinol, or combinationsthereof. In certain embodiments, the nanotubes are fabricated oftitania. In addition, other examples of suitable metal or metal alloysinclude, but are not limited to: stainless steels (e.g., 316, 316L or304), nickel-titanium alloys including shape memory or superelastictypes (e.g., nitinol or elastinite); inconel; noble metals includingcopper, silver, gold, platinum, palladium and iridium; refractory metalsincluding Molybdenum, Tungsten, Tantalum, Titanium, Rhenium, or Niobium;stainless steels alloyed with noble and/or refractory metals; magnesium;amorphous metals; plastically deformable metals (e.g., tantalum);nickel-based alloys (e.g., including platinum, gold and/or tantalumalloys); iron-based alloys (e.g., including platinum, gold and/ortantalum alloys); cobalt-based alloys (e.g., including platinum, goldand/or tantalum alloys); cobalt-chrome alloys (e.g., elgiloy);cobalt-chromium-nickel alloys (e.g., phynox); alloys of cobalt, nickel,chromium and molybdenum (e.g., MP35N or MP20N); cobalt-chromium-vanadiumalloys; cobalt-chromium-tungsten alloys; platinum-iridium alloys;platinum-tungsten alloys; magnesium alloys; titanium alloys (e.g., TiC,TiN); tantalum alloys (e.g., TaC, TaN); L605; bioabsorbable materials,including magnesium; or other biocompatible metals and/or alloysthereof. Preferably, the implantable frame comprises a self-expandingnickel titanium (NiTi) alloy material, stainless steel or acobalt-chromium alloy. The nickel titanium alloy sold under thetradename Nitinol.

In some embodiments, at least a subset of the plurality of nanofibers,nanotubes, or microwells further include an erodible capping film toprovide for delayed elution of the bioactive agent to the surroundingtissue upon placement of the implant in a subject. For example, thesurface or film will include a first subset of nanotubes including abioactive agent and a second subset of nanotubes including a bioactiveagent and capped with an erodible capping film. In such an example, thefirst subset of nanotubes lacking the erodible capping film will elutethe bioactive agent upon placement in the subject, thereby providingearly release of the bioactive agent. Over time, as the capping filmover the second subset of nanotubes erodes, the bioactive agent will bereleased. As a result, the combination of capped and uncapped structuresprovides for two elution profiles, a first early elution from theuncapped subset and a second later elution following erosion of thecapping film from the second capped subset.

In certain embodiments, the surface or film includes a plurality of atleast two of nanofibers, nanotubes, and microwells, wherein one of saidnanofibers, nanotubes, and microwells further include an erodiblecapping film to provide for delayed elution of said bioactive agent tothe surround tissue upon placement of the implant in a subject. Forexample, the surface or film will include a plurality nanotubes and aplurality of microwells, wherein include a bioactive agent and eitherthe nanotubes or microwells further include an erodible capping film. Insuch an example, the structure lacking the erodible capping film willelute the bioactive agent upon placement in the subject, therebyproviding early release of the bioactive agent. Over time, as thecapping film over the capped structure erodes, the bioactive agent willbe released. As a result the combination of capped and uncappedstructures provides for two elution profiles, a first early elution fromthe uncapped structures and a second later elution following erosion ofthe capping film from the second capped structure.

In general, the nanotubes or nanofibers are fabricated to have adiameter ranging from about 3 nm to about 300 nm, including about 10 nmto about 250 nm, about 20 nm to about 225 nm, about 30 nm to about 200nm, about 50 nm to about 190 nm, about 60 nm to about 180 nm, about 70nm to about 170 nm, about 80 nm to about 160 nm, and about 90 nm toabout 150 nm. In some embodiments, the nanofibers are fabricated at adensity greater than at least about 100,000,000 nanofibers per squarecentimeter or more, including at least about 200,000,000 nanofibers persquare centimeter, and at least about 300,000,000 nanofibers per squarecentimeter. In some embodiments the nanotubes are fabricated at adensity greater than at least about 10,000,000 nanotubes per squarecentimeter, including at least about 25,000,000 nanotubes per squarecentimeter, and at least about 50,000,000 nanotubes per squarecentimeter. In some embodiments, the nanofibers and nanotubes arefabricated at a density greater than at least about 25,000,000nanofibers per square centimeter or more, including at least about50,000,000 nanofibers per square centimeter, and at least about75,000,000 nanofibers per square centimeter, wherein the densityprovides for an extracellular matrix compatible tissue adhesive.

In general, the nanotubes or nanofibers are fabricated to have a lengthranging from about 1 μm to about 500 μm, including about 2 μm to about450 μm, about 3 μm to about 400 μm, about 4 μm to about 350 μm, about 5μm to about 300 μm, about 6 μm to about 250 μm, about 7 μm to about 200μm, about 8 μm to about 100 μm, about 9 μm to about 90 μm, about 10 μmto about 18 μm, about 11 μm to about 70 about 12 μm to about 60 μm,about 13 μm to about 50 μm, about 14 μm to about 40 μm, about 15 μm toabout 30 μm, and about 16 μm to about 20 μm. In an exemplary embodiment,the nanotubes have a length of about 10 μm.

In general, the nanotubes are fabricated to have pores range in diameterfrom about 3 nm to about 250 nm, including 4 nm to about 225 nm,including 5 nm to about 200 nm, including 6 nm to about 175 nm,including 7 nm to about 150 nm, including 8 nm to about 125 nm,including 9 nm to about 100 nm, including 10 nm to about 75 nm,including 11 nm to about 70 nm, including 12 nm to about 65 nm,including 13 nm to about 60 nm, including 14 nm to about 50 nm,including 15 nm to about 45 nm, about 20 nm to about 40 nm, about 22 nmto about 38 nm, about 24 nm to about 36 nm, about 26 nm to about 34 nm,about 28 nm to about 32 nm, and about 29 nm to about 31 nm. In anexemplary embodiment, the pores have in diameter of about 20 nm to about40 nm.

In general, the microwells are fabricated to have a diameter rangingfrom about 1 μm to about 150 μm, including about 2 μm to about 125 μm,about 3 μm to about 100 μm, about 4 μm to about 80 μm, about 5 μm toabout 60 μm, about 6 μm to about 50 μm, about 7 μm to about 40 m, about8 μm to about 30 μm, and about 7 μm to about 20 μm. In some embodiments,the microwells are fabricated to have a diameter ranging from about 1 μmto about 12 μm. In some embodiments, the microwells are fabricated at adensity greater than at least about 150,000 microwells per squarecentimeter or more, including at least about 200,000 microwells persquare centimeter, and at least about 300,000 microwells per squarecentimeter.

In general, the surface or film is fabricated to have a thicknessranging from about 1 μm to about 2.5 mm, including about 2 μm to about 2mm, about 3 μm to about 1.5 mm, about 3 μm to about 1 mm, about 4 μm toabout 750 μm, and about 5 μm to about 600 μm. In certain embodiments,the surface or film have a thickness ranging from about 1 μm to about200 μm, including about 3 μm to about 150 μm, about 4 μm to about 100μm, about 5 μm to about 80 μm, about 6 μm to about 70 μm, about 7 μm toabout 60 μm, about 8 μm to about 50 μm, about 9 μm to about 40 μm, andabout 10 μm to about 30 μm. In an exemplary embodiment, the surface orfilm has a thickness of about 150 μm.

In certain embodiments, the surface or film further includesadvantageous biological agents and additives to impart, for example,additional osteoinductive and osteoconductive properties to thesurface-modified implants. In further embodiments, the advantageousbiological agents and additives are added to the nanotubes, nanowires,or microwells for elution to the surrounding tissue upon placement ofthe implant in the patient. This may be particularly useful for implantsof the present invention that are bone implants. In an exemplaryembodiment, one or more biological agents or additives may be added tothe implant before implantation. The biological agents and additives maybe adsorbed onto and incorporated into the surface or film comprisingnanotubes, nanowires, or microwells, by dipping the implant into asolution or dispersion containing the agents and/or additives, or byother means recognized by those skilled in the art. In some embodiments,the nanotubes, nanowires, or microwells will release the adsorbedbiological agents and additives in a time-controlled fashion. In thisway, the therapeutic advantages imparted by the addition of biologicalagents and additives may be continued for an extended period of time. Itmay be desirable to include certain additives in the electrolytesolution used during the electrochemical anodization process in order toincrease the adsorptive properties of the nanotubes formed on thesurface-modified implant. For example, the inclusion of salts in theelectrolyte solution used during the electrochemical anodization processmay result in the incorporation of ionic substances into the nanotubes,nanowires, or microwells formed on the surface or film. The inclusion ofionic substances in the nanotubes, nanowires, or microwells may impartgreater adsorptive properties to the nanotubes due to the polarinteractions between the nanotubes, nanowires, or microwells containingionic substances and the biological agents and additives.

The biological agents or additives may be in a purified form, partiallypurified form, recombinant form, or any other form appropriate forinclusion in the surface-modified medical implant. It is desirable thatthe agents or additives be free of impurities and contaminants.Exemplary agents to facilitate cell adhesion and cell grow includelaminin, fibrin, fibronectin, proteoglycans, glycoproteins,glycosaminoglycans, chemotactic agents, and growth factors, and thelike.

For example, growth factors may be included in the surface-modifiedimplant or to the nanotubes for elution to the surrounding tissue uponplacement of the implant in the patent to encourage bone or tissuegrowth. Non-limiting examples of growth factors that may be included areplatelet derived growth factor (PDGF), transforming growth factor b(TGF-b), insulin-related growth factor-I (IGF-I), insulin-related growthfactor-II (IGF-II), fibroblast growth factor (FGF), beta-2-microglobulin(BDGF II), and bone morphogenetic factors. Bone morphogenetic factorsare growth factors whose activity is specific to bone tissue including,but not limited to, proteins of demineralized bone, demineralized bonematrix (DBM), and in particular bone protein (BP) or bone morphogeneticprotein (BMP). Osteoinductive factors such as fibronectin (FN),osteonectin (ON), endothelial cell growth factor (ECGF), cementumattachment extracts (CAE), ketanserin, human growth hormone (HGH),animal growth hormones, epidermal growth factor (EGF), interleukin-1(IL-1), human alpha thrombin, transforming growth factor (TGF-beta),insulin-like growth factor (IGF-1), platelet derived growth factors(PDGF), and fibroblast growth factors (FGF, bFGF, etc.) also may beincluded in the surface-modified implant.

Still other examples of biological agents and additives that may beincorporated in the nanotopography of the medical implant arebiocidal/biostatic sugars such as dextran and glucose; peptides; nucleicacid and amino acid sequences such as leptin antagonists, leptinreceptor antagonists, and antisense leptin nucleic acids; vitamins;inorganic elements; co-factors for protein synthesis; antibodytherapies, such as Herceptin®, Rituxan®, Myllotarg®, and Erbitux®;hormones; endocrine tissue or tissue fragments; synthesizers; enzymessuch as collagenase, peptidases, and oxidases; polymer cell scaffoldswith parenchymal cells; angiogenic agents; antigenic agents;cytoskeletal agents; cartilage fragments; living cells such aschondrocytes, bone marrow cells, mesenchymal stem cells, naturalextracts, genetically engineered living cells, or otherwise modifiedliving cells; autogenous tissues such as blood, serum, soft tissue, andbone marrow; bioadhesives; periodontal ligament chemotactic factor(PDLGF); somatotropin; bone digestors; antitumor agents andchemotherapeutics such as cis-platinum, ifosfamide, methotrexate, anddoxorubicin hydrochloride; immuno-suppressants; permeation enhancerssuch as fatty acid esters including laureate, myristate, and stearatemonoesters of polyethylene glycol; bisphosphonates such as alendronate,clodronate, etidronate, ibandronate,(3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (APD),dichloromethylene bisphosphonate, aminobisphosphonatezolendronate, andpamidronate; pain killers and anti-inflammatories such as non-steroidalanti-inflammatory drugs (NSAID) like ketorolac tromethamine, lidocainehydrochloride, bipivacaine hydrochloride, and ibuprofen; antibiotics andantiretroviral drugs such as tetracycline, vancomycin, cephalosporin,erythromycin, bacitracin, neomycin, penicillin, polymycin B, biomycin,chloromycetin, streptomycin, cefazolin, ampicillin, azactam, tobramycin,clindamycin, gentamicin, and aminoglycocides such as tobramycin andgentamicin; and salts such as strontium salt, fluoride salt, magnesiumsalt, and sodium salt.

Examples of antimicrobial agents include, but are not limited to,tobramycin, amoxicillin, amoxicillin/clavulanate, amphotericin B,ampicillin, ampicillin/sulbactam, atovaquone, azithromycin, cefazolin,cefepime, cefotaxime, cefotetan, cefpodoxime, ceftazidime, ceftizoxime,ceftriaxone, cefuroxime, cefuroxime axetil, cephalexin, chloramphenicol,clotrimazole, ciprofloxacin, clarithromycin, clindamycin, dapsone,dicloxacillin, doxycycline, erythromycin, fluconazole, foscarnet,ganciclovir, atifloxacin, imipenem/cilastatin, isoniazid, itraconazole,ketoconazole, metronidazole, nafcillin, nafcillin, nystatin, penicillin,penicillin G, pentamidine, piperacillin/tazobactam, rifampin,quinupristin-dalfopristin, ticarcillin/clavulanate,trimethoprim/sulfamethoxazole, valacyclovir, vancomycin, mafenide,silver sulfadiazine, mupirocin, nystatin, triamcinolone/nystatin,clotrimazole/betamethasone, clotrimazole, ketoconazole, butoconazole,miconazole, and tioconazole.

Antiangiogenic agents include, but are not limited to, interferon-α,COX-2 inhibitors, integrin antagonists, angiostatin, endostatin,thrombospondin-1, vitaxin, celecoxib, rofecoxib, JTE-522, EMD-121974,and D-2163, FGFR kinase inhibitors, EGFR kinase inhibitors, VEGFR kinaseinhibitors, matrix metalloproteinase inhibitors, marmiastat,prinomastat, BMS275291, BAY12-9566, neovastat, rhuMAb VEGF, SU5416,SU6668, ZD6474, CP-547, CP-632, ZD4190, thalidomide and thalidomideanalogues, squalamine, celecoxib, ZD6126, TNP-470, and otherangiogenesis inhibitor drugs.

In general, the bioactive agent eluting nanotubes, nanowires, ormicrowells will elute the bioactive agent to the surrounding tissue uponplacement of the implant in the patient for a period raging from about 2minutes to about 3 months or more, including 5 minutes to about 14weeks, such as about 24 hours, 72 hours, about 3 days, about 7 days,about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8weeks, about 12 weeks, or more.

In further embodiments, in which is desirable to modulate the releasekinetics of the bioactive agents that is eluted from the nanotubes,nanowires, or microwells a suitable synthetic or natural polymer iscombined with the bioactive agent prior to or at the same time thenanotubes, nanowires, or microwells are loaded with the bioactive agent.Suitable synthetic and natural polymers include, but are not limited to,biodegradable or bioerodible polymers, such aspoly(DL-lactide-co-glycolide) (PLGA), poly(DL-lactide-co-ε-caprolactone)(DLPLCL), or poly(ε-caprolactone) (PCL), collagen, gelatin, agarose, andother natural biodegradable materials.

The mineral nanotubes, nanowires, or microwells surfaces with or withoutadhesion-promoting peptides and/or other biological agents, can becompacted and/or structured and used alone to form an implant.Alternatively, a structured substrate can be coated with a compositioncomprising the nanotubes, nanowires, or microwells with or withoutadhesion-promoting peptides. Substrates include any conventionalsubstrates for medical implants or for other types of implants known inthe art.

Also provided is a method of treating a patient in need of a medicalimplant comprising the steps of selecting the medical implant whereinthe implant comprises nanotube, nanowires, or microwells coated surfaceand placing the implant into the patient. Exemplary implants include,orthopedic implants, dental implants, cardiovascular implants, such as apacemaker, neurological implants, neurovascular implants,gastrointestinal implants, muscular implants, ocular implants, and thelike. In this embodiment of the invention the term “selecting” means,for example, purchasing, choosing, or providing the implant rather thanpreparing the implant.

The method of the present invention can be used for both human clinicalmedicine and veterinary applications. Thus, the patient can be a humanor, in the case of veterinary applications, can be a laboratory,agricultural, domestic, or wild animal. The present invention can beapplied to animals including, but not limited to, humans, laboratoryanimals such as monkeys and chimpanzees, domestic animals such as dogsand cats, agricultural animals such as cows, horses, pigs, sheep, goats,and wild animals in captivity such as bears, pandas, lions, tigers,leopards, elephants, zebras, giraffes, gorillas, dolphins, and whales.

In another embodiment, a method for enhancing osseointegration of anorthopedic implant is provided. The method comprises the steps ofselecting the orthopedic implant wherein the implant comprises nanotube,nanowires, or microwells coated surface and placing the implant into apatient. In this embodiment of the invention the term “selecting” means,for example, purchasing, choosing, or providing the implant rather thanpreparing the implant. The patient can be a human or, in the case ofveterinary applications, can be a laboratory, agricultural, domestic, orwild animal.

Enhancement of osseointegration is increased osseointegration comparedto that obtained with conventional implant materials. Enhancedosseointegration can be demonstrated by increased osteoblast adhesion,increased osteoblast proliferation, increased calcium deposition, enzymeactivity assays, or by any other art-recognized technique used to detectosseointegration.

In yet another embodiment a method of preparing a medical implant isprovided. The method comprises the step of forming a compositioncomprising nanotubes, nanowires, or microwells. The method can furthercomprise the step of coating a substrate with the nanotube, nanowire, ormicrowell-containing composition. The composition formed can be acomposition containing the nanotubes, nanowires, or microwells alone, ananocomposite composition, a nanocomposite composition containing anadhesion-promoting peptide, or any other composition containingnanotubes, nanowires, or microwells that is suitable for use inaccordance with the present invention.

Kits

Kits for use in connection with the subject invention are also provided.The above-described surface or film comprising nanofibers, nanotubes, ormicrowells, comprising a bioactive agent for elution to the surroundingtissue upon placement in a subject, as well as medical implantsincluding the surface or film, can be provided in kits, with suitableinstructions in order to conduct the methods as described above. The kitwill normally contain in separate containers the nanotubes or materialsnecessary for fabricating the nanotubular coating on a surface.Instructions (e.g., written, tape, VCR, CD-ROM, etc.) for carrying outthe methods usually will be included in the kit. The kit can alsocontain, depending on the particular method, other packaged reagents andmaterials (i.e. buffers and the like).

The instructions are generally recorded on a suitable recording medium.For example, the instructions may be printed on a substrate, such aspaper or plastic, etc. As such, the instructions may be present in thekits as a package insert, in the labeling of the container of the kit orcomponents thereof (e.g., associated with the packaging orsubpackaging), etc. In other embodiments, the instructions are presentas an electronic storage data file present on a suitable computerreadable storage medium, e.g., CD-ROM, diskette, etc, including the samemedium on which the program is presented.

In yet other embodiments, the instructions are not themselves present inthe kit, but means for obtaining the instructions from a remote source,e.g. via the Internet, are provided. An example of this embodiment is akit that includes a web address where the instructions can be viewedfrom or from where the instructions can be downloaded.

Still further, the kit may be one in which the instructions are obtainedare downloaded from a remote source, as in the Internet or world wideweb. Some form of access security or identification protocol may be usedto limit access to those entitled to use the subject invention. As withthe instructions, the means for obtaining the instructions and/orprogramming is generally recorded on a suitable recording medium.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Methods and Materials

The following methods and materials were used in the Examples below.

Fabrication of Titania Nanotubular Surfaces

Titania nanotubular surfaces were fabricated using an anodizationprocess described elsewhere (Mor et al., Advanced Functional Materials2005, 15:1291-96; Varghese et al., Journal of Materials Research 2003,18:156-155). In brief, titanium foils (Alfa Aesar) of thickness 0.25 mmand 99.8% purity were used to fabricate titania nanotubes. Theelectrolyte consisted of 0.5 vol % hydrofluoric acid (J. T. Baker) inwater, and a platinum (Alfa Aesar) electrode served as a cathode.Anodization was performed at a constant voltage of 20V for 45 mins. Thesamples were cleaned using deionized water after completing theanodization process. The nanotubes were then sintered at 500° C. in dryoxygen as it is known that these ambients influence the phasetransformation of titania. The surface morphologies of the samples werestudied using a Sirion Scanning Electron Microscope (SEM).

Isolation and Culture of Marrow Stromal Cells (MSC)

Male Lewis rats were obtained from the Laboratory of Animal ResourceCenter (LARC) at University of California, San Francisco. The animalswere euthanized according to an IACUC approved protocol. Limbs wereremoved aseptically and placed in cold phosphate buffer solution (PBS)in 50 ml falcon tubes. Bones were dissected from the soft tissues undera cell culture hood. Metaphyseal ends of the bones were removed to allowaccess to the marrow cavity. The contents of marrow cavity were flushedout using a 25-gauge needle attached to a 10 ml syringe containingalpha-modified MEM (αMEM) supplemented with 10% FBS and 1% penn/strep.The flushed cell suspension was then filtered through a 70 μm nylonstrainer. In order to seed cells on nanotubular surfaces (1 cm×1 cm),surfaces were adhered to the bottom of 12-well plates with medical-gradesilicone (Dow) and cured overnight. The plates were then placed underultraviolet light in a biological hood for 30 mins. Before seeding thecells, the surfaces were soaked in 70% ethanol for 30 minutes forsterilization. The surfaces were then washed twice with warm PBS and thecells were plated at a density of 5×106 per well. On day 4 of culture,half of the media was removed and replaced with fresh αMEM supplementedwith 10% FBS and 1% penn/strep. On day 7 of culture, all media wasremoved and cells were supplied with the complete media. The completeMedia includes αMEM supplemented with 10% FBS, 1% Penn/Strep,Dexamethasone (10-8M final concentration), Ascorbic acid (50 μg/mlfinal) and beta-glycerol phosphate (8 mmol final). Media was thenchanged every two days for the duration of the experiment. Cell responsewas investigated in two stages; (a) cell adhesion and proliferation upto 7 days after the initial culture phase and (b) cell differentiationfor up to 3 weeks after providing complete media (i.e. after 7 days ofinitial culturing). Commercially available titanium and tissue culturepolystyrene were used as standards.

MSC Adhesion and Proliferation

MSC adhesion was investigated 1 day after seeding the cells andproliferation was investigated after days 4 and 7. The adhered andproliferated cells were quantified by trypsinizing the cells on surfacesand counting them using a hemacytometer.

Cell Viability

The cell viability was investigated 4 days after seeding the cells usinga commercially available MTT assay (Sigma). The MTT method is simple,accurate and yields reproducible results. The key component is(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) or MTT.Solutions of MTT, dissolved in medium or balanced salt solutions withoutphenol red, are yellowish in color. Mitochondrial dehydrogenases ofviable cells cleave the tetrazolium ring, yielding purple formazancrystals. The standard protocol provided with the MTT kit was followed.The resulting purple solution was spectrophotometrically measured at 570nm using a spectrophotometer. An increase or decrease in cell numberresulted in a concomitant change in the amount of formazan formed,indicating the degree of cytotoxicity caused by the surfaces.

Calcein Staining

After 7 days of culture, the cells on the surfaces were stained withcalcein. The polyanionic dye calcein is well retained within live cells,thus producing an intense uniform green fluorescence in live cells. Thesurfaces were washed twice with PBS before staining. They were incubatedin 2 μM solution of calcein for 30-45 mins. The surfaces were imagedusing a fluorescence microscope after washing with PBS.

Total Intracellular Protein Content

Total protein content of MSCs is extremely important since it is theindication of healthy growth and normal cell response on nanotubularenvironments. Thus, the amount of protein produced by cells was measuredup to 3 weeks of culture after providing complete media. In order torelease the intracellular protein, the adhered cells on the substrateswere lysed in deionized water using a standard four cycle freeze-thawmethod. The resulting lysate solution was then used for analysis. Thetotal protein content was determined by a BCA (bicinchoninic acid) assaykit (Pierce) and the absorbance of the solution was measured using aspectrophotometer at a wavelength of 570 nm. The absorbance was thenconverted to protein content using an albumin standard curve todetermine the amount of intracellular protein.

Alkaline Phosphatase Activity

ALP activity is an important parameter to access the normalfunctionality of cells on a surface; hence the activity was measured upto 3 weeks after providing complete media. The resulting lysate solutionwas used to measure the ALP activity using a commercially availablecolorimetric assay (Teco). The absorbance of the solution was measuredusing a spectrophotometer at a wavelength of 590 nm. The absorbance wasthen converted to concentration using ALP standard and all the data wasnormalized with total protein content to account for changes in numberof cells present on each surface.

Analysis of Calcium Content

The calcium content was measured up to 3 weeks in culture using acolorimetric assay (Teco). After the lysate was removed; the surfaceswere soaked overnight in 6N HCl solution to dissolve the depositedcalcium. The calcium solution was then reacted with assay reagents andthe absorbance of the solution was measured photometrically at 570 nm.The absorbance was then converted to concentration using calciumstandards and all the data was normalized with total protein content toaccount for changes in the number of cells present on each surface.

Extracellular Matrix Production

Calcium and phosphorus are the primary components of bone matrix. Ifcells have differentiated on the surfaces, they will begin to depositbone matrix. In order to detect the presence of calcium and phosphoruson our surfaces, the samples were air dried for XPS analysis. XPS is asurface sensitive technique and detects trace levels of elements presenton the surface. Survey spectra were collected from 0 to 1100 eV using aSSI S-Probe Monochromatized XPS Spectrometer with Al-Kα-X-ray small spotsource (1486.6 eV) and multichannel detector with a pass energy of 160eV. Data for percent atomic composition and atomic ratios for depositedcalcium and phosphorous on alumina surfaces for up to three weeks ofculture were calculated from the survey scans using the manufacturersupplied software.

Cell Morphology

Cell morphology on nanotubular and control surfaces was examined usingSEM. The surfaces were imaged after 1, 4 and 7 days of culture toinvestigate the adhesion and proliferation stage; and after 1, 2 and 3weeks of culture after providing complete media. Prior to imaging, thecells were fixed and dehydrated. The surfaces were rinsed twice in PBSand then soaked in the primary fixative of 3% glutaraldehyde (Sigma),0.1M of sodium cacodylate (Polysciences), and 0.1M sucrose (Sigma, St.Louis Mo.) for 45 minutes. The surfaces were subjected to twofive-minute washes with a buffer containing 0.1M sodium cacodylate and0.1M sucrose. The cells were then dehydrated by replacing the bufferwith increasing concentrations of ethanol (35, 50, 70, 95 and 100%) forten minutes each. Further, the cells were dried by replacing ethanol byhexamethyldisilazane (HMDS) (Polysciences) for 10 minutes. The HMDS wasremoved, and the surfaces were air dried for 30 minutes. SEM imaging wasconducted on the Sirion Scanning Electron Microscope at voltages rangingfrom 10-20 kV after the surfaces were sputter coated in gold. Thesputter coater was set at current of 20 mA and pressure of 0.05 mbar fortwenty seconds to deposit a 10 nm layer of gold.

In Vivo Immune Response of Titania Nanotubular Surfaces

Two male Lewis rats, about 225 gm, were anesthetized in an induction boxwith 3% isoflurane and then kept on 1.5% isoflurane on a fitted nosemask during the course of the surgery. Animals were clipped and preppedfollowing aseptic techniques described in the guidelines by IACUC. A 1cm midline incision was made in the scruff region of the neck. Thesample implant discs (5 mm diameter and 1 mm thick) were sterilized byautoclaving. The skin layer was detached from the muscle layer forming apocket on each side of the incision, were the surfaces were placed. Twosamples were implanted in each animal making a total of 4 samples (2nanotubular titania and 2 titania control). The skin layer was thensutured with 4.0 nylon suture. The animals were allowed to recoverbefore being returned to the animal facility. They were monitored everyday for the first week and twice a week thereafter until 4 weeks. Nylonstitches were removed after a week. After 4 weeks, the animals wereanesthetized as described above. A midline incision was made in thescruff region of the neck. Skin layer was lifted to expose the implantswhich were then retrieved together with the surrounding tissue. Theanimals were then sacrificed by cardiac removal.

Directly after euthanasia, retrieved implants with the surroundingtissues were fixed in 4% phosphate buffered formaldehyde solution.Subsequently, the specimens were dehydrated and embedded in Spurr'sembedding media. After polymerization, sections of 300 μm in thicknesscontaining tissue/implant interface were obtained using a slow-speeddiamond saw (Buehler Isomet saw). The sections were then grounded using400 and 600 grit sand paper on a wheel grinder (Buehler Ecomet IIIgrinder) to a final thickness of 50 μm and stained with haematoxylin andeosin.

Statistical Analysis

Each experiment was reconfirmed at least three times using cells fromdifferent marrow stromal preparations. All the results were analyzedusing analysis of variance (ANOVA). Statistical significance wasconsidered at p<0.05.

Filling of Nanotubes

The nanotubes were filled via a simplified lyophilization method(Foraker et al. Pharm Res 20(1):110-6 (2003); Salonen et al. J ControlRelease 108(2-3):362-74 (2005)). In brief, 100 mg/ml of solutions of BSAin PBS (pH 7.1) and LYS in sodium acetate buffer (50 mM, pH 4.5) wereprepared. Titania nanotube surfaces (0.5 cm×0.5 cm) were cleaned withdeionized water prior to loading of BSA and LYS. 1 μL of proteinsolution was pipetted onto the nanotube surface and gently spread toensure even coverage. The surfaces were then allowed to dry under vacuumat room temperature for 2 hours. After drying, the loading step wasrepeated until the appropriate amount of protein was present in thenanotube array. In this way the surfaces were loaded with 200, 400 and800 μg of protein. After the final drying step, the surfaces were rinsedquickly by pipetting 500 μl of PBS over the surface to remove any excessprotein on the surface. The rinse solutions were collected and storedfor further analysis. Nanotube surfaces adsorbed with the sameconcentration of protein were used as controls. The adsorption wascarried out by incubating the surfaces for 20 minutes followed byrinsing with PBS.

X-Ray Photoelectron Spectroscopy

To evaluate the differences in amounts of protein in nanotubes, X-rayphotoelectron spectroscopy (XPS) analysis was carried out. XPS is asurface sensitive technique which can detect changes in surfacecomposition for up to 2-20 atomic layers, depending on the material.Since the nanotubes are approximately 400 nm long, XPS can be used toverify the differences in surface concentrations due to differentamounts of BSA and LYS loaded. The loaded nanotube surfaces along withadsorbed surfaces were mounted on an XPS stage. Three spots per samplewere analyzed. The analysis was conducted on a SSI S-ProbeMonochromatized XPS Spectrometer which uses an Al-Kα-X-ray source(1486.6 eV) with an Omni Focus III small area lens and multichanneldetector. A concentric hemispherical analyzer (CHA) was operated in theconstant analyzer transmission mode to measure the binding energies ofemitted photoelectrons. The binding energy scale was calibrated by theAu4f_(7/2) peak at 83.9 eV, and the linearity was verified by theCu3p_(1/2) and Cu2p_(3/2) peaks at 76.5 and 932.5 eV respectively.Survey spectra were collected from 0 to 1100 eV with pass energy of 160eV, and high-resolution C1s spectra were collected for each elementdetected with pass energy of 10 eV. Survey and high resolution spectrawere collected at a 65° take off angle, defined as the angle spanned bythe electron path to the analyzer and the sample surface. All spectrawere referenced by setting the hydrocarbon C1s peak to 285.0 eV tocompensate for residual charging effects. Data for percent atomiccomposition and atomic ratios were calculated using the manufacturersupplied sensitivity factor.

Release from Nanotubes

In order to release the proteins from the nanotubes, the surfaces wereimmersed in 500 μl of PBS in a 24-well plate at room temperature withorbital shaking at 70 rpm. 200 μl of samples were taken after specificintervals of time to determine the release kinetics. Samples werecollected periodically for up to 120 minutes. The solution was replacedwith 200 μl of fresh PBS every time the samples were taken. The sampleswere analyzed for protein content using a commercially availableMicro-BCA assay kit and the concentration was adjusted for dilutions dueto replacement of fresh PBS.

Example 1 Fabrication of Nanotubular Surface Coated Implants

The results show development of nanotubular surfaces that can be appliedto existing implants, thereby providing a strategy that can be appliedquite readily in the clinical environment. The approach to achieving anoptimal material nanoarchitecture uses a simple anodization technique tofabricate vertically oriented, immobilized, high-aspect ratio titaniananotube arrays. FIG. 1 shows an SEM image of titania nanotubes withpore size of approximately 80 nm and length of 400 nm prepared usinganodization voltage of 20V for 20 mins. It can be seen that the nanotubearray is substantially uniform over the substrate surface. There is aprecise correlation between the anodization voltage and pore size, thusby varying the voltage and anodization time, substrates with differentsize scales can be fabricated (Mor et al., Journal of Materials Research2003, 18(11):2588-93; Rougraff et al., J Bone Joint Surg Am 2002,84-A(6):921-9). The large surface area of the nanotube-array structureand the ability to precisely tune pore size, wall-thickness, andnanotube length to optimize biotemplating properties are among the manydesirable properties of this architecture to use them for orthopedicapplications.

The fabricated nanotubular titania surfaces were seeded with marrowstromal cells obtained from male Lewis rats. Bone marrow extractscontaining osteoprogenitors combined with various matrices have beenshown to accelerate and enhance bone formation within osseous defectswhen compared with the matrix alone (Rougraff et al., J Bone Joint SurgAm 2002, 84-A(6):921-9; Tiedeman et al., Orthopedics 1995,18(12):1153-8). MSCs contain a pluripotent population of cells capableof differentiating along multiple mesenchymal lineages (e.g., bone(Haynesworth et al., Bone 1992, 13(1):81-8; Prockop et al., Science1997, 276(5309):71-4), ligament (Altman et al., Faseb J 2002;16(2):270-2), adipose (Beresford et al., J Cell Sci 1992; 102 (Pt2):341-51), cartilage (Wakitani et al., J Bone Joint Surg Am 1994;76(4):579-92) and muscle tissue (Seshi et al., Blood Cells Mol Dis 2000;26(3):234-46)). Because tissue culture techniques allow the isolationand ex vivo expansion of this cell population from animals, these cellsrepresent an ideal osteogenic cell source to be used to evaluate theirinteraction with nanostructured surfaces. The ability of MSCs to inducebone formation in vivo is believed to be due to the interaction ofosteoprogenitors present within the cell populations with osteoinductivefactors, such as bone morphogenetic proteins and various growth factorsand cytokines, which cause them to differentiate into bone-forming cellsi.e. osteoblasts, which will then eventually form bone matrix.Commercially available pure titanium and tissue culture polystyrene wereused as controls.

Significant attachment to the surfaces is necessary in order for MSCs tospread and differentiate. By determining the initial attachment of MSCsonto the nanotubular surfaces, we can examine the correlation of cellattachment and physical and mechanical properties of the scaffold.Contact and interactions between cells will eventually affect thedifferentiation process. Thus, cell adhesion and proliferation wasinvestigated on nanotubular titania surfaces; and was compared to thatfrom titanium and polystyrene. FIG. 2, panel a, shows the results of MSCadhesion after 1 day and proliferation after 4 and 7 days of culturingthe cells. As, expected, polystyrene supported highest cell adhesion andproliferation. However, the results indicated a 40% increase in thenumber of cells present on nanotubular titania surfaces compared to flattitania surfaces (p<0.05) after 7 days of culture. The results show thattopographical cues at nanoscale level present on the nanotubular titaniasurfaces promote cell adhesion and proliferation.

Higher adhesion on the surface does not necessarily suggest that thecells are viable and functional. Therefore, the cell viability was alsoassessed using the MTT assay. FIG. 2, panel b, shows the absorbancevalues obtained for cells adhered to surfaces for 4 days. The resultsshow that the cells are viable on nanotubular titania surfaces as wellas on titanium and polystyrene surfaces. The absorbance values fornanotubular and titanium surfaces are similar to that obtained frompolystyrene. Polystyrene is commonly used as a positive control for cellculture. Hence the similarity between the absorbance values shows thatthe cells are healthy and viable on all three surfaces. After 7 days ofculture (just before providing the complete media), the cells werestained with calcein. Calcein staining is fluorescence based stainingmethod for assessing cell viability. It is a faster, less expensive andmore sensitive indicator of cytotoxic events. Live cells aredistinguished by the presence of intracellular esterase activity,determined by the enzymatic conversion of the virtually nonfluorescentcell-permeant calcein to the intensely fluorescence calcein. Thepolyanionic dye calcein is well retained within the live cells,producing uniform green fluorescence in live cells. FIG. 3, panels a andb, shows fluorescence microscopy images of MSCs on titanium andnanotubular surfaces respectively stained with calcein. The images showthat the cells are viable on these surfaces after 7 days of culture.Furthermore, closer inspection of cells on nanotubular surfaces revealsthe formation of clusters, which is a normal phenotypic behavior ofMSCs. This behavior is absent on flat titanium surfaces, showing thatthe nanotubular surfaces are providing a more favorable microenvironmentfor MSCs.

MSC morphology on titanium and nanotubular titania surfaces wasinvestigated using SEM. FIG. 4 shows SEM images of MSCs after 1, 4 and 7days of culture on titanium and nanotubular surfaces. As expected, thecells are spherical after day 1 on both titanium and nanotubularsurfaces (FIG. 4, panels a and b, respectively). After 4 days ofculture, the MSCs still show a spherical morphology on titanium surfaces(FIG. 4, panel c); however they show a spreading morphology onnanotubular surfaces (FIG. 4, panel d). By day 7, the MSCs on titaniumsurfaces are still isolated with minimal spreading (FIG. 4, panel e),whereas the MSCs on nanotubular surfaces have formed a networkindicative of cell-cell communication (FIG. 4, panel f). These resultsshow that the MSCs are able to spread faster on nanotubular surfaces ascompared to titanium surfaces within 7 days of culture. Highmagnification SEM images were taken after 7 days on nanotubular surfacesto visualize the cell extensions. FIG. 4, panels g and h show highmagnification SEM images of an MSC extension probing the nanotubulararchitecture. The length of the extension is many times greater then thecell diameter. These extensions help the cell anchor itself to thenanotubular structure. By doing so, the cells can adhere and spread onthe surface, resulting in enhanced long term differentiation.

After 7 days of culture on titanium and nanotubular titania surfaces,the MSCs were provided with complete media to initiate differentiationand matrix deposition. Alkaline phosphatase activity (ALP) activity wasmeasured for up to 3 weeks of culture after providing complete media.The ALP levels are age dependent; however the levels are elevated duringthe period of active bone growth, and thus were measured. A colorimetricassay was used to measure the ALP levels. FIG. 5, panel a shows the ALPactivity normalized with total protein content to account for differencein number of cells present on each surface. The cells present onnanotubular surfaces show higher ALP levels compare to those on titaniumsurfaces. There is approximately a 50% increase in ALP levels onnanotubular surfaces after 3 weeks of culture (p<0.05).

As the cells differentiate, they begin to deposit bone matrix on thesurface. The bone matrix predominantly consists of calcium phosphate.Thus, the amount of calcium and phosphorous on the surfaces can bemeasured using X-ray photoelectron spectroscopy (XPS). XPS is a surfacesensitive technique and detect presence of trace amount of elements onthe surface. Table 1 shows the ratios of atomic concentrations ofcalcium and phosphorous to that of titanium obtained by survey scans oftitanium and nanotubular surfaces for up to 3 weeks of culture. Theresults show a steady increase in the amounts of calcium and phosphorouson nanotubular surfaces, compared to a negligible increase on titaniumsurfaces. This corresponds to greater bone matrix deposition onnanotubular surfaces as compared to titanium surfaces. Furthermore, thecalcium deposited on all surfaces was dissolved in hydrochloric acid andits concentration was measured using a colorimetric assay. Calciumreacts with cresolphthalein complex one in 8-hydroxyquinoline to form apurple color which was then measured photometrically. FIG. 5, panel bshows the calcium concentration normalized with respect to total proteincontent to account for the difference in cell number on each surface.These results closely correlate with the results obtained from XPS.Again, there is approximately 50% increase in calcium content onnanotubular surfaces compared to titanium surfaces for up to 3 weeks ofculture (p<0.05). These results show that nanotubular surfaces provide afavorable interface for MSC differentiation and matrix production.

TABLE 1 Ca/Ti P/Ti Titanium Nanotubes Titanium Nanotubes Week 1 0.310.32 0.37 0.41 Week 2 0.32 0.56 0.42 0.78 Week 3 0.34 1.21 0.44 1.65

The morphology of MSCs during the differentiation phase on nanotubularsurfaces was investigated using SEM. FIG. 6 shows SEM images of MSCs onnanotubular surfaces for up to 3 weeks of culture. The cells show aspreading morphology and network formation on the surface after 1 weekof culture (FIG. 6, panel a). High magnification SEM images show thepresence of granular material on the nanotubular surface (FIG. 6, panelb). After 2 weeks, the SEM images show that the whole surface is coveredwith a network of well spread cells (FIG. 6 panel c). A close look atthe areas surrounding the cells confirms that the nanopores are beingfilled in with matrix (FIG. 6, panel d). After 3 weeks of culture, theSEM images show that the whole surface is completely covered with bothcells and mineralized matrix components (FIG. 6, panel e). Again, a highmagnification SEM image of the area around the cells shows that thenanotubular structures are completely filled with a porous material. Asdiscussed earlier, XPS analysis shows that this deposited materialpredominantly consisted of calcium and phosphorous, important bonematrix constituents.

It is crucial that any new biomaterial used for orthopedic applicationsmust demonstrate appropriate biocompatibility. Thus the in vivobiocompatibility of titanium and nanotubular surfaces was investigatedby implanting discs of titanium and nanotubular titania subcutaneously.After 4 weeks, the implants were retrieved and biocompatibility wasevaluated by histological analysis of the tissue surrounding theimplant. FIG. 7 shows a light microscopy image of sections of healthytissue and the tissue surrounding the implant stained with haematoxylinand eosin. FIG. 7, panel a, shows histology sections of healthy tissue.There is no fibrous scar tissue present in the tissues surrounding thetitanium implant and comparable to healthy tissue (FIG. 7, panel b).Titanium is known to be biocompatible, and therefore should not causeany undesirable immune response in vivo. FIG. 7, panel c, shows lightmicroscopy images of tissue sections surrounding the nanotubular titaniaimplant. Similar to titanium, there is no fibrous scar tissue formationaround the implant. The tissue appears to be healthy and normal. Thus,these preliminary in vivo results show that the nanotubular surfaces donot cause any adverse immune response under in vivo conditions.

The development of nanostructured platforms based on novel metal-oxidefilms can provide insight into cell-material interactions for thedevelopment of improved implant surfaces. The results provided hereinshow that nanotubular titania surfaces provide a favorable template forbone cell growth and differentiation. Nanotubular titania surfaces werefabricated by a simple anodization process and were used to investigateshort term and long term performance of MSCs. The results show thatthese surfaces supported higher cell adhesion, proliferation andviability up to 7 days of culture when compared to titanium surfaces.Cells cultured on nanotubular surfaces demonstrated higher ALP activity.Furthermore, the calcium and phosphorous concentrations were 50% higheron these surfaces showing that matrix deposition was upregulated onnanotubular surfaces. Moreover, the nanotubular surfaces do not causeadverse immune response under in vivo conditions. Thus, the results showthat osteoblast activity can be significantly enhanced using controllednanotopographies. Therefore, incorporation of such nanoarchitectures onimplant surfaces will further facilitate the culture and maintenance ofdifferentiated cell states, and promote long-term osseointegration

Example 2 Fabrication of Drug Eluting Nanotubular Surface CoatedImplants

It was next determined whether the nanotubes may be filled withantibiotics that would ward off infection immediately after implantation(e.g., gentamicin, chlorhexidine diacetate, ciprofloxacin) or withgrowth factors or therapeutic proteins (e.g., TGF-β, IGF, BMP) that willhelp to stimulate cellular differentiation and bone repair processes. Inthis study, bovine serum albumin (BSA) and lysozyme (LYS) were used asmodel proteins to investigate their loading and release efficienciesfrom nanotube architectures. BSA is a larger molecule with a netnegative charge at neutral pH compared to LYS which is smaller in sizewith a net positive charge at neutral pH (Table 2).

TABLE 2 Molecular Isoelectric Weight Point Net charge (KDa) pI @ pH 7.0Bovine Serum Albumin 67 4.7 −18 Lysozyme 14 11 +7 

Before the release studies were performed, it was important to evaluatethe loading efficiency of the proteins on the nanotube surface. Theconcentrations of the original and the rinse solutions were measuredusing a commercially available Micro-BCA assay kit. The loadingefficiency was expressed as percentage of loaded protein after washing.The loading efficiency was calculated by the following equation:

$\begin{matrix}{\eta = \frac{C_{o} - C_{r}}{C_{o}}} & (1)\end{matrix}$where η: loading efficiency

C_(o): protein concentration in the original solution

C_(r): protein concentration in the rinse solution

FIG. 8 shows loading efficiencies for nanotubes loaded with 200, 400 and800 μg of both BSA and LYS. The results show that approximately 60-80%of protein is retained in the nanotubes after washing regardless ofinitial loading.

To evaluate the differences in amounts of protein in nanotubes, X-rayphotoelectron spectroscopy (XPS) analysis was carried out. Table 3 showsthe N/C ratios computed from XPS survey scans. LYS and BSA adsorbedsurfaces were used as controls. There is a steady increase in N/C ratioswith increasing amounts of protein loaded into the nanotubes. A moreprecise way to characterize protein on the surface is to determine thefraction of C—N and N—C═O peaks in overall C1s peak. The C—N and N—C═Opeaks are characteristic of proteins which are at a shift of 0.8 eV and1.8 eV respectively from the C—C peak (285 eV). Thus, high resolutionC1s scans were taken and the peak fit analysis software provided withthe XPS instrument was used to determine % of C—N and N—C═O in theoverall C1s peak (Table 3). A convolution of Gaussian components wasassumed for all the peaks. There is an increase in the intensity for theC—C, C—N and N—C═O peaks with increasing amounts of proteins loaded intothe nanotubes. However, the survey and high resolution C1s scans forsurfaces adsorbed with BSA and LYS show significantly lower proteinconcentrations on the surface. These results show that the nanotubes canbe successfully loaded with measured amounts of proteins using thetechnique described here.

TABLE 3 Bovine Serum Albumin Lysozyme Adsorbed 200 mg 400 mg 800 mgAdsorbed 200 mg 400 mg 800 mg N/C 0.123 0.188 0.233 0.268 0.215 0.2450.268 0.297 C—C 0.81 0.71 0.51 0.40 0.73 0.53 0.32 0.21 C—N 0.11 0.180.24 0.20 0.22 0.29 0.35 0.40 N—C═O 0.08 0.11 0.25 0.40 0.03 0.18 0.310.39

FIG. 9 shows the release data obtained from nanotubes loaded with 200,400 and 800 μg of BSA and LYS. The amount of protein eluted is expressedin terms of fraction of total protein released. The results show thattwo different proteins, a larger negative molecule (BSA, FIG. 9, PanelsA, B and C) and a smaller positive molecule (LYS; FIG. 9, Panels D, Eand F), can be easily released from the nanotubes. Further, the releasekinetics can be altered by changing the amount of protein loaded. Table4 shows the time points at which all the protein is released from thenanotubes.

As expected, there is slower and sustained release from the nanotubesloaded with a higher amount of protein compared to those loaded withlower amounts of protein. Also, the data shows that LYS release from thenanotubes is much slower compared to that of BSA. It is thought thatthis is due to the difference between the negatively and positivelycharged proteins interacting with surface charge of the nanotubeinterface. The surfaces of most metal oxide films are inherently chargedas a consequence of the equilibration of charged crystalline latticedefects within the surface. Depending on the net concentration oflattice defects the surface may be positively or negative charged. Thesurface of titania nanotubes consists of terminal hydroxyl groups whichresults in mild negative charge on the surface. Thus, the fact that therelease of LYS which is positively charged is much slower compared tothat of BSA which is negatively charged may be due to a strongerelectrostatic interaction between the LYS and the titania surface.

TABLE 4 Bovine Serum Albumin Lysozyme 200 μg 25 36 400 μg 50 85 800 μg65 110

This study shows that titania nanotubes can be easily fabricated with ananodization process and. These nanotubes can also be optionally loadedwith drugs or biological agents such as proteins. Moreover, the releaseor elution of the drugs or biological agents from the nanotubes can becontrolled by varying the tube length, diameter and wall thickness.Here, we have shown that the release rates of BSA and LYS can becontrolled by varying their loading into the nanotubes themselves theamounts loaded into nanotubes. Furthermore, by changing the nanotubediameter, wall thickness and length, the release kinetics can be alteredfor specific drugs in order to achieve sustained release of the drugover a period of time. Thus, these nanotubular surfaces have potentialapplications in orthopedics, specifically for implants where fasterosseointegration is desired along with controlled release of drugs suchas antibiotics or growth factors.

What is claimed is:
 1. A method comprising administering to a subject acomposition that comprises a surface or film comprising a verticallyoriented array of a plurality of nanotubes or microwells, wherein abioactive agent is filled into the nanotubes or microwells themselves,wherein a first end of the array of the plurality of filled nanotubes ormicrowells is in contact with the surface or film, and wherein theplurality of nanotubes or microwells is capped with a polymeric erodiblecapping film to provide for delayed elution of the bioactive agent fromwithin the nanotubes or microwells to the surrounding tissue uponplacement in a subject and erosion of the capping film.
 2. The method ofclaim 1, wherein the subject is a human.
 3. The method of claim 1,wherein the subject is a laboratory, agricultural, domestic or wildanimal.
 4. The method of claim 1, wherein the composition is anorthopedic implant, a dental implant, a cardiovascular implant, aneurological implant, a neurovascular implant, a gastrointestinalimplant, a muscular implant, or an ocular implant.
 5. The method ofclaim 4, wherein the composition is configured for enhancingosseointegration of the orthopedic implant.
 6. The method of claim 1,wherein the composition is a patch for localized delivery of saidbioactive agent to a soft tissue.
 7. The method of claim 1, wherein saidsurface or film unfurls in the presence of a hydrating liquid.
 8. Themethod of claim 1, wherein said surface or film further comprises acovalently attached bioactive agent.
 9. The method of claim 1, whereinsaid surface or film is comprised of poly(DL-lactide-co-glycolide)(PLGA), poly(DL-lactide-co-ε-caprolactone) (DLPLCL),poly(ε-caprolactone) (PCL), gelatin, agarose, poly(methyl methacrylate),galatin/ε-caprolactone, collagen-GAG, collagen, fibrin, PLA, PGA,PLA-PGA co-polymers, poly(anhydrides), poly(hydroxy acids), poly(orthoesters), poly(propylfumerates), poly(caprolactones),poly(hydroxyvalerate), polyamides, polyamino acids, polyacetals,biodegradable polycyanoacrylates, biodegradable polyurethanes andpolysaccharides, polypyrrole, polyanilines, polythiophene, polystyrene,polyesters, non-biodegradable polyurethanes, polyureas, poly(ethylenevinyl acetate), polypropylene, polymethacrylate, polyethylene,polycarbonates, poly(ethylene oxide), co-polymers of the above, mixturesof the above, and adducts of the above, or combinations thereof.
 10. Themethod of claim 1, wherein said nanotubes range in length from about 1μm to about 500 μm.
 11. The method of claim 1, wherein said nanotubesrange in diameter from about 3 nm to about 300 nm.
 12. The method ofclaim 11, wherein said surface or film comprises nanotubes at a densitygreater than 10,000,000 nanotubes per square centimeter.
 13. The methodof claim 12, wherein said surface or film comprises nanotubes at adensity greater than 25,000,000 nanotubes per square centimeter, whereinsaid density provides for an extracellular matrix compatible tissueadhesive.
 14. The method of claim 1, wherein said nanotubes have a porediameter range from about 3 nm to about 250 nm.
 15. The method of claim1, wherein said surface or film ranges in thickness from about 1 μm toabout 2.5 mm.
 16. The method of claim 1, wherein said microwells rangein diameter from about 1 μm to about 150 μm.
 17. The method of claim 16,wherein said surface or film comprises microwells at a density greaterthan 150,000 microwells per square centimeter.
 18. The method of claim1, wherein said nanotubes or microwells further comprise an agent tofacilitate cell adhesion and cell growth selected from the groupconsisting of laminin, fibrin, fibronectin, proteoglycans,glycoproteins, glycosaminoglycans, chemotactic agents, and growthfactors.
 19. The method of claim 1, wherein said bioactive agent isselected from a polypeptide, growth factor, a steroid agent, an antibodytherapy, an antibody fragment, a DNA, an RNA, and siRNA, anantimicrobial agent, an antibiotic, an antiretroviral drug, ananti-inflammatory compound, an antitumor agent, anti-angiogeneic agent,and a chemotherapeutic agent.
 20. The method of claim 1, wherein saidsurface or film further comprises cells.
 21. The method of claim 20,wherein said cell is a stem cell, a retinal progenitor cell, a cardiacprogenitor cell, an osteoprogenitor cell, or a neuronal progenitor cell.